A Critical Analysis of the Role of SNARE Protein SEC22B in Antigen Cross-Presentation

S. Julia Wu; Yashar S. Niknafs; Stephanie H. Kim; Katherine Oravecz-Wilson; Cynthia Zajac; Tomomi Toubai; Yaping Sun; Jayendra Prasad; Daniel Peltier; Hideaki Fujiwara; Israel Hedig; Nathan D. Mathewson; Rami Khoriaty; David Ginsburg; Pavan Reddy

doi:10.1016/j.celrep.2017.06.013

A Critical Analysis of the Role of SNARE Protein SEC22B in Antigen Cross-Presentation

S. Julia Wu, Yashar S. Niknafs, Stephanie H. Kim, Katherine Oravecz-Wilson, Cynthia Zajac, Tomomi Toubai, Yaping Sun, Jayendra Prasad, Daniel Peltier, Hideaki Fujiwara, Israel Hedig, Nathan D. Mathewson, Rami Khoriaty, David Ginsburg, Pavan Reddy

Research output: Contribution to journal › Article › peer-review

35 Citations (Scopus)

Abstract

Cross-presentation initiates immune responses against tumors and viral infections by presenting extracellular antigen on MHC I to activate CD8⁺ T cell-mediated cytotoxicity. In vitro studies in dendritic cells (DCs) established SNARE protein SEC22B as a specific regulator of cross-presentation. However, the in vivo contribution of SEC22B to cross-presentation has not been tested. To address this, we generated DC-specific Sec22b knockout (CD11c-Cre Sec22b^fl/fl) mice. Contrary to the paradigm, SEC22B-deficient DCs efficiently cross-present both in vivo and in vitro. Although in vitro small hairpin RNA (shRNA)-mediated Sec22b silencing in bone-marrow-derived dendritic cells (BMDCs) reduced cross-presentation, treatment of SEC22B-deficient BMDCs with the same shRNA produced a similar defect, suggesting the Sec22b shRNA modulates cross-presentation through off-target effects. RNA sequencing of Sec22b shRNA-treated SEC22B-deficient BMDCs demonstrated several changes in the transcriptome. Our data demonstrate that contrary to the accepted model, SEC22B is not necessary for cross-presentation, cautioning against extrapolating phenotypes from knockdown studies alone.

Original language	English
Pages (from-to)	2645-2656
Number of pages	12
Journal	Cell Reports
Volume	19
Issue number	13
DOIs	https://doi.org/10.1016/j.celrep.2017.06.013
Publication status	Published - Jun 27 2017
Externally published	Yes

Keywords

RNA-seq
SEC22B
SNARE protein
cross-presentation
dendritic cell
off-target effect

ASJC Scopus subject areas

Biochemistry, Genetics and Molecular Biology(all)

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10.1016/j.celrep.2017.06.013

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Wu, S. J., Niknafs, Y. S., Kim, S. H., Oravecz-Wilson, K., Zajac, C., Toubai, T., Sun, Y., Prasad, J., Peltier, D., Fujiwara, H., Hedig, I., Mathewson, N. D., Khoriaty, R., Ginsburg, D., & Reddy, P. (2017). A Critical Analysis of the Role of SNARE Protein SEC22B in Antigen Cross-Presentation. Cell Reports, 19(13), 2645-2656. https://doi.org/10.1016/j.celrep.2017.06.013

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title = "A Critical Analysis of the Role of SNARE Protein SEC22B in Antigen Cross-Presentation",

abstract = "Cross-presentation initiates immune responses against tumors and viral infections by presenting extracellular antigen on MHC I to activate CD8+ T cell-mediated cytotoxicity. In vitro studies in dendritic cells (DCs) established SNARE protein SEC22B as a specific regulator of cross-presentation. However, the in vivo contribution of SEC22B to cross-presentation has not been tested. To address this, we generated DC-specific Sec22b knockout (CD11c-Cre Sec22bfl/fl) mice. Contrary to the paradigm, SEC22B-deficient DCs efficiently cross-present both in vivo and in vitro. Although in vitro small hairpin RNA (shRNA)-mediated Sec22b silencing in bone-marrow-derived dendritic cells (BMDCs) reduced cross-presentation, treatment of SEC22B-deficient BMDCs with the same shRNA produced a similar defect, suggesting the Sec22b shRNA modulates cross-presentation through off-target effects. RNA sequencing of Sec22b shRNA-treated SEC22B-deficient BMDCs demonstrated several changes in the transcriptome. Our data demonstrate that contrary to the accepted model, SEC22B is not necessary for cross-presentation, cautioning against extrapolating phenotypes from knockdown studies alone.",

keywords = "RNA-seq, SEC22B, SNARE protein, cross-presentation, dendritic cell, off-target effect",

author = "Wu, {S. Julia} and Niknafs, {Yashar S.} and Kim, {Stephanie H.} and Katherine Oravecz-Wilson and Cynthia Zajac and Tomomi Toubai and Yaping Sun and Jayendra Prasad and Daniel Peltier and Hideaki Fujiwara and Israel Hedig and Mathewson, {Nathan D.} and Rami Khoriaty and David Ginsburg and Pavan Reddy",

note = "Funding Information: This work was supported by the US NIH grants HL-128046, HL-090775, CA-173878, CA-203542 and the Herman and Dorothy Miller Fund. We acknowledge use of the Vector Core, Flow Cytometry Core, and DNA Sequencing Core of the University of Michigan's Biomedical Research Core Facilities for preparation of reagents, use of equipment, processing of samples, and generation of data. Publisher Copyright: {\textcopyright} 2017 The Author(s)",

year = "2017",

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doi = "10.1016/j.celrep.2017.06.013",

language = "English",

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T1 - A Critical Analysis of the Role of SNARE Protein SEC22B in Antigen Cross-Presentation

AU - Wu, S. Julia

AU - Niknafs, Yashar S.

AU - Kim, Stephanie H.

AU - Oravecz-Wilson, Katherine

AU - Zajac, Cynthia

AU - Toubai, Tomomi

AU - Sun, Yaping

AU - Prasad, Jayendra

AU - Peltier, Daniel

AU - Fujiwara, Hideaki

AU - Hedig, Israel

AU - Mathewson, Nathan D.

AU - Khoriaty, Rami

AU - Ginsburg, David

AU - Reddy, Pavan

N1 - Funding Information: This work was supported by the US NIH grants HL-128046, HL-090775, CA-173878, CA-203542 and the Herman and Dorothy Miller Fund. We acknowledge use of the Vector Core, Flow Cytometry Core, and DNA Sequencing Core of the University of Michigan's Biomedical Research Core Facilities for preparation of reagents, use of equipment, processing of samples, and generation of data. Publisher Copyright: © 2017 The Author(s)

PY - 2017/6/27

Y1 - 2017/6/27

N2 - Cross-presentation initiates immune responses against tumors and viral infections by presenting extracellular antigen on MHC I to activate CD8+ T cell-mediated cytotoxicity. In vitro studies in dendritic cells (DCs) established SNARE protein SEC22B as a specific regulator of cross-presentation. However, the in vivo contribution of SEC22B to cross-presentation has not been tested. To address this, we generated DC-specific Sec22b knockout (CD11c-Cre Sec22bfl/fl) mice. Contrary to the paradigm, SEC22B-deficient DCs efficiently cross-present both in vivo and in vitro. Although in vitro small hairpin RNA (shRNA)-mediated Sec22b silencing in bone-marrow-derived dendritic cells (BMDCs) reduced cross-presentation, treatment of SEC22B-deficient BMDCs with the same shRNA produced a similar defect, suggesting the Sec22b shRNA modulates cross-presentation through off-target effects. RNA sequencing of Sec22b shRNA-treated SEC22B-deficient BMDCs demonstrated several changes in the transcriptome. Our data demonstrate that contrary to the accepted model, SEC22B is not necessary for cross-presentation, cautioning against extrapolating phenotypes from knockdown studies alone.

AB - Cross-presentation initiates immune responses against tumors and viral infections by presenting extracellular antigen on MHC I to activate CD8+ T cell-mediated cytotoxicity. In vitro studies in dendritic cells (DCs) established SNARE protein SEC22B as a specific regulator of cross-presentation. However, the in vivo contribution of SEC22B to cross-presentation has not been tested. To address this, we generated DC-specific Sec22b knockout (CD11c-Cre Sec22bfl/fl) mice. Contrary to the paradigm, SEC22B-deficient DCs efficiently cross-present both in vivo and in vitro. Although in vitro small hairpin RNA (shRNA)-mediated Sec22b silencing in bone-marrow-derived dendritic cells (BMDCs) reduced cross-presentation, treatment of SEC22B-deficient BMDCs with the same shRNA produced a similar defect, suggesting the Sec22b shRNA modulates cross-presentation through off-target effects. RNA sequencing of Sec22b shRNA-treated SEC22B-deficient BMDCs demonstrated several changes in the transcriptome. Our data demonstrate that contrary to the accepted model, SEC22B is not necessary for cross-presentation, cautioning against extrapolating phenotypes from knockdown studies alone.

KW - RNA-seq

KW - SEC22B

KW - SNARE protein

KW - cross-presentation

KW - dendritic cell

KW - off-target effect

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SN - 2211-1247

VL - 19

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EP - 2656

JO - Cell Reports

JF - Cell Reports

IS - 13

ER -

A Critical Analysis of the Role of SNARE Protein SEC22B in Antigen Cross-Presentation

Abstract

Keywords

ASJC Scopus subject areas

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